PROJECT SUMMARY Despite recent therapeutic advances, metastatic melanoma remains a disease with poor prognosis. Patients with primary melanomas that are clinically and histologically similar at the time of initial diagnosis often have vastly different outcomes, from patients who are cured after initial surgical resection to those that develop recurrence(s), metastatic progression, and eventually die. Such highly variable outcomes suggest underlying biological differences in patient tumors (cell-intrinsic) or the patients themselves (cell-extrinsic, e.g. immune response). Recent studies suggest that early tumorigenic events can reflect a melanoma's potential to spread. Conceptually, if such molecular alterations can be robustly measured at the time of melanoma diagnosis, they may be useful prognostic markers. Moreover, some of these markers may also be functional drivers of disease progression, thus their study may yield novel insights into melanoma biology and new therapeutic targets. We hypothesize that altered gene expression can predict patient outcome and, moreover, that some prognostic biomarkers are functional drivers of melanoma progression. Our group and others have observed that expression of various mRNA and microRNA (miRNA) may have prognostic value for patients with primary melanoma. We propose to examine the expression of a panel of ~230 mRNA/miRNA previously associated to poor outcomes in melanoma in a multi-institutional cohort of primary melanoma patients (n = 1000, stages IIA- IIIB at diagnosis) with extensive clinical follow-up. Using this expression data, we propose to develop and validate a refined tissue-based, molecular prognostic mRNA/miRNA signature for stages IIA to IIIB melanomas (Aims 1 and 2). In addition, we will investigate if candidate prognostic genes (as defined by each of the P01 projects) can be functional mediators of the aggressive phenotype. We propose to perform in vivo pooled library-based functional screens to examine the effects of modulation of candidate prognostic genes on tumor growth and metastatic potential of melanoma cells (Aim 3.1). Moreover, we will investigate cellular properties underlying the effects of functionally relevant candidate genes identified in in vivo screens (Aim 3.2). A molecular signature that, at initial diagnosis, can reliably predict outcomes for primary melanoma patients could transform clinical management of these individuals, informing selection of higher-risk patients for increased surveillance and/or adjuvant therapy. The hope is that better melanoma patient management will lead to improved outcomes, such as prolonging patient survival or reducing morbidity and mortality. In addition, the described functional studies will reveal candidate genes (from all projects of the P01) that contribute to melanoma progression and metastasis, which might open new therapeutic avenues against this devastating disease. !